title: Properties of mutationally altered RNA polymerases II of Drosophila.
abstract: We tested and compared several in vitro properties of wild type and mutant RNA polymerases II from Drosophila melanogaster, using several different mutants of a single X-linked genetic locus, RpIIC4 (Greenleaf, A. L., Weeks, J. R., Voelker, R. A., Ohnishi, S., and Dickson, B. (1980) Cell 21, 785-792); the mutants tested included the original amanitin-resistant mutant, C4, which is nonconditional, plus the temperature-sensitive mutants A9, C20, E28, and 1Fb40. Using a tritium-labeled amanitin derivative, we demonstrated that C4 polymerase has a reduced binding affinity for amanitin. The C4 polymerase was as stable to thermal denaturation as the wild type enzyme, and the two enzymes had similar specific activities, ionic strength and Mn2+ requirements, and apparent Km values for UTP and GTP when assayed in the presence of Mn2+. However, with Mg2+ as the divalent cation, C4 polymerase was less active than wild type and had 2-fold higher apparent Km values for UTP and GTP. Three of the temperature-sensitive mutants, A9, C20, and E28, were derived from the amanitin-resistant mutant C4; the polymerase II activities from these mutants displayed resistance to alpha-amanitin in vitro identical with that of the C4 enzyme. C20, E28, and 1Fb40 polymerases were markedly less stable to thermal denaturation in vitro than wild type polymerase. The results presented indicate that the mutations at the RNA polymerase locus (RpIIC4-) directly alter the structure of the enzyme, providing conclusive evidence that the locus is a structural gene for a polymerase II subunit.
mesh_terms: D000818:Animals; D002413:Cations, Divalent; D012321:DNA-Directed RNA Polymerases / Q000235:genetics*; D004331:Drosophila melanogaster / Q000201:enzymology* / Q000235:genetics; D004625:Embryo, Nonmammalian / Q000201:enzymology; D007700:Kinetics; D009154:Mutation; D009994:Osmolar Concentration; D012319:RNA Polymerase II / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism
chemical_list: D002413:Cations, Divalent; D012319:RNA Polymerase II; D012321:DNA-Directed RNA Polymerases